Methods for treating mental fatigue

ABSTRACT

CH3-CO-NH-CH(-CH2-COOH)-CO-NH-CH(-(CH2)2-COOH)-COOH   AND N-ACETYL-B-L-ASPARTYL-L-GLUTAMIC ACID   CH3-CO-NH-CH(-COOH)-CH2-CO-NH-CH(-COOH)-(CH2)2-COOH   AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM WHICH ARE USED FOR THE TREATMENT OF MENTAL FATIGUE AND REALTED SYNDROMES. A PROCESS FOR THE PREPARATION OF N-ACETYL-A-LASPARTYL-L-GLUTAMIC ACID AND N-ACETYL-B-L-ASPARTYL-LGLUTAMIC ACID IS ALSO DISCLOSED.

United States Patent 01 c Patented Apr. 25, 1972 US. Cl. 424-319 8Claims ABSTRACT OF THE DISCLOSURE N-acetyl-a-aspartyl-L-glutamic acidC1130 ONHCH-C ONH-C O OH CH2C OOH (CH2) z-COOH and N-acetyl-B--aspartyl-L-glutamic acid 0 H3CO-NHCHC O OH CH2CONHCH-C O OH Han-000Eand pharmaceutical compositions containing them which are used for thetreatment of mental fatigue and related syndromes. A process for thepreparation of N-acetyl-a-L- aspartyl-L-glutamic acid andN-acetyl-B-L-aspartyl-L- glutamic acid is also disclosed.

The present application is a continuation-in-part of application Ser.No. 805,902, filed Mar. 10, 1969, which is a continuation of applicationSer. No. 528,355, filed Feb. 18, 1966, both now abandoned.

The present invention relates to the following substances:N-acetyl-a-L-aspartyl-L-glutamic acid Hz-COOH (CH2)2COOH (I) andN-acetyl-B-L-aspartyl-L-glutamic acid:

CH3CONHCHC 0 011 CHzCO-NHCH-C O OH H2)2C0OH (II) and to pharmaceuticalcompositions containing the acids; a process for preparing said acids,and to substances related to said acids.

The object of the present invention is the preparation of pharmaceuticalcompositions which may be used for the treatment of mental fatigue andrelated syndromes. A further object of the invention consists in aprocess for the preparation of the substances N-acetyl-a-L-aspartyl-L-glutamic acid and N-acetyI- S-L-aspartyl-L-glutamic acid.

A further object of the invention consists in preparation of thefollowing substances which relate to N-acetyla-L-aspartyl-L-glutamicacid and to N-acetyl-B-L-aspartyl- L-glutamic acid, inasmuch as theyconstitute the stages of the synthetic preparation of the latter.

B-Methyl ester of N-acetyl-L-aspartic acid:

o Hz-COOCH; (III) B-Ethyl ester of N-acetyl-L-aspartic acid:

CH3CONHCHC 0 OH BIZ-C O O C2115 N-acetyl-methyl-a-L-aspartyl-diethyl-L-glutamate OH3CONHCH-C ONH-CHC o 0 02115 CHz-COOGHACHQPCOOC H (Va)N-acetyl-ethyl-u-L-aspartyl-diethyl-L-glutamate:

(VII) (VIII) The synthesis process of acids I and II consistsessentially in the previous preparation of glutamates Va and Vb (forN-acetyl-u-L-glutamic acid I and of glutamates VI and VIII (forN-acetyl-fi-L-glutamic acid (II) which glutamates are subjectedthereafter to a controlled alkaline hydrolysis followed by acidificationwith mineral acids and vacuum concentration. Alternatively, triesters Vaand Vb can be subjected to a treatment with resin Dowex50W X4 (strongacid) (a trademark registered by the Dow Chemical (10., U.S.A.) andsuccessive lyophilization, whereby N-acetyl-a-L-aspartyl-L-glutamic acid(I) is obtained which can be purified by crystallization. The controlledalkaline hydrolysis of N-acetyl-fl-L-aspartyl-diethyl-L-glutamate (VI)leads to N-acetyl-fl-L-aspartyl-L- glutamic acid (H), whose isolationfrom the reaction mixture is carried out by treating the alkalinesolution (coming from the hydrolysis) with mineral acids andconcentrating in a vacuum.

At the stage of preparation ofN-acetyl-methyl-a-aspartyl-diethyl-L-glutamate (Va) and ofN-acetyl-ethyl-a- L-aspartyl-diethyl-L-glutamate (Vb) there is carriedout the condensation of a beta-ester of N-acetyl-L-aspartic acid, forexample the ,B-methyl ester (III) of N-acetyl-L- aspartic acid or thefl-ethyl ester (IV) of N-acetyl-L- aspartic acid, with a diester ofL-glutamic acid. The 3- methyl ester (III) of N-acetyl-L-aspartic acidis prepared from beta-methyl ester of L-aspartic acid by reacting withacetic anhydride.

The condensation of the fi-methyl ester (III) of N- acetyl-L-asparticacid or of ,B-ethyl ester (IV) of N-acetylaspartic acid with a diesterof L-glntamic acid is carried out, according to the invention, throughthe preparation of mixed anhydrides, which are obtained by reacting anorganic base salt (for example, triethylamine) of B-methyl ester (III)of N-acetyl-L-aspartic acid or fl-ethyl ester (IV) ofN-acctyl-L-aspartic acid with an alkyl chlorocarbonate (for example,ethyl, isopropyl, or isobutyl chlorocarbonate). The mixed anhydrideswithout being isolated are directly condensed in suitable conditionswith a diester of L-glutamic acid to give, in the case ofdiethyl-L-glutamate, N-acetyl-methyl u-L-aspartyl-diethyl- L-glutamate(Va) or, respectively, N-acetyl-ethyl-a-L- aspartyl-diethyl-L-glutamate(Vb).

Alternatively, another preferred method of synthesis of the estersN-acetyl-methyl-a-L-aspartyl-diethybL- glutamate (Va) andN-acetyl-ethyl-a-L-aspartyl-diethyl- L-glutamate (Vb) is given by thedirect condensation of the fi-methyl ester (III) of N-acetyl-L-asparticacid or of the fl-ethyl ester ('IV) of N-acetyl-L-aspartic acid with adiester of L-glutamic acid by means of dicyclohexylcarbodiimide in asuitable solvent such as methylene chloride.

The stage of preparation of N-acetyl-B-L-aspartyl-diethyl-glutamate iscarried out according to the invention by condensing the anhydride ofN-acetyl-L-aspartic acid with a diester of L-glutamic acid such as thediethyl ester. This reaction leads to a mixture ofN-acetyl-a-L-aspartyldiethyl-L-glutamate and its beta-isomer (VI).

The separation of these two isomers is carried out by taking advantageof the higher acidity of the carboxyl group ofN-acetyl-B-L-aspartyl-diethyl L glutamate (VII); practically by means ofa fractional extraction with diluted solutions of alkaline carbonates,of a solution of the mixture of the two isomer in ethyl acetate. Byacidifying the first fractions the separation of N-acetyl-fl-L-aspartyl-diethyl-L-glutamate (VI) occurs.

Lastly, the stage of preparation of N-acetyl-methyl-fl-L-aspartyldiethyl-L-glutamate (VIII) comprises the formation of a mixedanhydride, which can be obtained by reacting the salt of methylN-acetyl-a-L-aspartate (VII) (in turn obtained by acetylatingmethyl-a-aspartate) with an organic base such as triethylamine and analkyl chlorocarbonate e.g. ethyl, isopropyl or isobutyl chlorocarbonatein a suitable solvent.

The mixed anhydride (without being isolated) is directly treated in thereaction mixture with diethyl-L-glutamate, to giveN-acetylmethyl-B-aspartyl-diethyl-L-glutamate (VIII).

The following examples illustrate the process of preparation of thecompounds. Of course variations to the process as indicated in theexamples can easily be made by experts in the art without departing fromthe scope of the invention. In the examples temperatures are given in C.In each example an individual process for obtaining the substanceinvolved as well as the stage of the entire process for reachingN-acetyl-a-L-aspartyl-L-glutamic acid (I) orN-acetyl-fl-L-aspartyl-L-glutamic acid (II), as above discussed, isgiven.

EXAMPLE 1 fi-rnethyl ester of N-acetyl-L-aspartic acid (III) 147 gramsof fl-methyl-L-aspartate is treated with 200 ml. of acetic acidanhydride for a period of one hour at 80-90".

The reaction mixture is cooled to 50 and the anhydride excess isdistilled otf at 1.5 mm. The residue is dissolved in 300 ml. absoluteethanol and the obtained solution is evaporated at 30 and 1.5 mm. ofpressure.

The latter operation is repeated 3 times, and the raw product, dissolvedin 70 ml. absolute ethanol, is crystallined by diluting with 200 ml.anhydrous ethyl ether and 200 ml. petroleum ether (B.P. 30-50).

After standing some hours at crystals of fi-methyl ester ofN-acetyl-L-aspartic acid (M.P. 144-5), M1 +9 (c.'=10 in absoluteethanol) are obtained.

A alysis-Calm. for C7H11O5N (percent): C, 44.44; H, 5.86. Found(percent): C, 44.56; H, 6.08.

EXAMPLE 2 N-acetyl-ethyl-a-L-aspartyl diethyl-L-glutamate (Vb) 40.6grams of N-acetyl-fi-ethyl-L-aspartate (IV) are dissolved in 400 ml.toluene and 150 ml. anhydrous chloroform. To the solution thus obtained,28.4 ml. triethylamine is added and the mixture is cooled to 5 C.Thereafter, 26 ml. isobutylchlorocarbonate is dropped slowly into thesolution; and after 15 minutes, there is added, little by little aprecooled (to -5) solution of 90 gramsdiethyl-L-glutamate-para-toluene-sulphonate and 34 ml. of triethylaminein an anhydrous mixture of 70' ml. toluene and 200 ml. chloroform.

Once the addition is completed, the temperature is allowed to rise to 20under stirring for 6 hours. Thereafter, the reaction mixture is washedwith 100 ml. 2 N HCl, 100 ml. sodium bicarbonate in 10% aqueoussolution, and lastly with 100 ml. water. The organic phase is dried withanhydrous sodium sulphate and the solvent is stripped at 60 and 15 mm.The oily residue is dissolved in ml. absolute alcohol and the solutionobtained is diluted with 300 ml. anhydrous ether. The mixture isfiltered by a folded filter and there is added 400 ml. petroleum ether(B.P. 30-50").

After standing for 12 hours at 0, crystals ofN-acetylethyl-aL-aspartyl-diethyl-L-glutamate are formed. Melting point94-5 [a] :=+24. (c.=5 in absolute ethanol).

AnalySis.Calcd. for C H O N (percent): C, 52.57; H, 7.27. Found(percent): C, 52.46; H, 7.22.

EXAMPLE 3 N-acetyl-methyl e-L-aspartyl diethyl-L-glutamate (Va) 56.7grams of N-acetyl-B-methyl L aspartate (III), dissolved in 500 ml.methylene chloride is treated with a solution of 112.5 gramsdiethyl-L-glutamate-para-toluensulphonate and 42 ml. triethylamine in250 ml. methyl ene chloride. Thereafter, a solution of 62 gramsdicyclohexylcarbodiimide in 300 ml. methylene chloride is added understirring, and the mixture is stirred for 6 hours at 22; thereafter, thereaction mixture is acidified with 3 ml. glacial acetic acid, is stirredfor 30 minutes more at 22, and then left to stand for 12 hours.

The mixture is vacuum filtered, and the filtrate is distilled at 15 mm.;the residue is solubilized with 1800 ml. ethyl acetate and the obtainedsolution, after being washed with ml. distilled water, is dried overanhydrous sodium sulphate and evaporated at 15 mm.

The oily residue is dissolved in 50 ml. absolute boiling ethanol, iscooled and diluted with 250 ml. anhydrous ethyl ether. The solution isfiltered through a folded filter, 300 ml. petroleum ether (B.P. 30-50")is added to the filtrate and the solution is left to stand at 0 for 12hours.

Thus, N-acetyl-methyl-a-L-aspartyl-diethyl L glutamate is obtained,melting point 122-4" and [a] 24 (c.=5 in water).

Analysis.-Calcd. for C H O N (percent): C, 51.33; H, 7.00. Found(percent): C, 51.37; H, 7.08.

EXAMPLE 4 N-acetyl-oz-L-aspartyl-L-glutamic acid (I) 73 grams N-acetyl-ethyl-e-L-aspartyl-diethyl-L-glutamate (Vb) are dissolved in 1130ml. N NaOH. The solution is kept for 2 hours at 20; then it is pouredinto a suitable chromatographic column prepared with 1400 grams resinDowex 50W X4 (strong acid) [trademark Dow Chemical Co.] 100-200 mesh, inH+ ionic form, moisture 65-72%.

The column is eluted with 3000 ml. distilled water and the eluate(sharply acid) is lyophilized.

The lyophilized solid crystallizes after dissolution in 300 ml.anhydrous boiling acetone.

Crystals of N-acetyl-a-L-aspartyl-L-glutamic acid are obtained, meltingpoint 171-5 with decomposition, and [ot] =-27 .5 (c.=2 in Water).

Analysis.-Calcd. for cnH gogNg (percent): C, 43.42; H, 5.30. Found(percent): C, 43.36; H, 5.46.

EXAMPLE 5 N-acetyl-fi-L-aspartyl-diethyl-L-glutamate (VI) 157 gramsN-acetyl-L-aspartic acid anhydride are suspended in 800 ml. ethylacetate and poured slowly into a solution of 203 gramsdiethyl-L-glutamate in 1000 ml. ethyl acetate. Thus, a solution isobtained which, after standing at 20 for 12 hours, is washedsuccessively with 200 ml. N HCl and 200 ml. water. The solution is thenextracted with 6 GO-mL-portions of a 12% aqueous solution of monohydratesodium carbonate.

The pool of alkaline solutions is acidified to a pH of 2.5 with 5 N HClto obtain a precipitate of N-acetyl-fi- L-aspartyl-diethyl-L-glutamate,which is purified by crystallization from water. The pure product has amelting point of l4950 and [a] '=-46 (c.= in water). Analysis.Calcd. forC H O N (percent): C, 49.99; H, 6.71. Found (percent): C, 49.78; H,6.54.

Example 6 N-aeetyl-fi-L-aspartyl-L-glutamic acid (II) 40 gramsN-acetyl-,B-L-aspartyl-diethyl-L-glutamate are dissolved in 400 ml. NNaOH; the solution is kept for 90 minutes at 22, then it is acidified topH 2 with 5 N HCl. The solution is lyophilized to obtain a dry residuewhich is dissolved in 150 ml. absolute boiling ethanol. The coolsolution is filtered through a folded filter, and the solvent isevaporated at 20 and 0.01 mm.

The dry residue is crystallized from acetone; thus, N- acetyl B Laspartyl L glutamic acid is obtained, melting point 103-5" (withdecomposition) and [a] 37 .5 (c.=2 in water).

Analysis.Calcd. for C H O N (percent): C, 43.42; H, 5.30. Found(percent): C, 43.46; H, 5.44.

EXAMPLE 7 m-methyl ester of N-acetyl-L-aspartic acid (VII) 30 gramsa-methyl-L-asparate are dissolved in 100 ml. distilled water; thetemperature of the solution is raised to 90, and 100 ml. acetic acidanhydride is added rapidly.

The solution is cooled to 20, 100 ml. more acetic acid anhydride isadded, and the mixture is stirred at 20 for 6 hours.

The solvent is evaporated at 30 and 0.01 mm., and the residue iscrystallized from acetone-petroleum ether, The pure ester VII isobtained, melting point 1212 and [a] =12 (c.=5 in absolute ethanol).

Analysis.Calcd. for C7H1105N (percent): C, 44.44; H, 5.86. Found(percent): C, 44.37; H, 5.90.

EXAMPLE 8 N-acetyl-methyl-B-L-aspartyl-diethyl- L-glutamate (VIII) Asolution of 37.8 grams N-acetyl-a-methyl-L-aspartate (VII) in 400 ml.anhydrous toluene, 150 ml. anhydrous chloroform and 28.4 ml.triethylamine, is cooled to 5 and is added with 26 ml.isobutylchlorocarbonate. After 15 minutes, a solution (precooled to 5)of 57.4 grams diethyl-L-glutamate hydrochloride in 70 ml. anhydroustoluene, 200 ml. anhydrous chloroform and 34 ml. triethylamine isdropped slowly. The temperature is then raised to 20, and the mixture isstirred for 6 hours, at this temperature, then it is washed successivelywith 100 ml. 2 N HCl, 100 ml. sodium bicarbonate in aqueous solution andwith 100 ml. distilled water.

The organic phase is dried with sodium sulphate and the solvents areremoved at 60 and mm.

The residues obtained by crystallization from mixtures of absolutealcohol and petroleum ether, yields the triester VIII, melting point1035 and [a] =-36 .2 (c.=4 in 95% ethanol).

Analysis.Calcd. for C H O N (percent): C, 51.33; H, 7.00. Found(percent): C, 51.13; H, 6.86.

The substances N-acetyl-u-L-aspartyl-L-glutamic acid andN-acetyl-B-L-aspartyl-L-glutamic acid may be combined in compositionssuitable for the treatment of man and animals to improve mentalefficiency or for the treatment of mental fatigue and related syndromes,which compositions comprise the above acids either singly or in mixturesthereof as active ingredient and pharmaceutical excipients known for theproduction of formulations suitable for oral or parenteraladministration. Suitable excipients are, for example, water, calciumcarbonate, lactose, starches, talcum, magnesium stearate, gelatin,vegetable oils and other known medicinal excipients. The pharmaceuticalpreparations may be, for example, a solid pharmaceutical composition inshaped dosage unit for oral ingestion, i.e., tablets or capsules, or thepreparations may be in liquid form, solutions, suspensions or emulsions.The above acids, either singly or in mixtures thereof, can be combinedwith an inert, pharmaceutically acceptable liquid carrier to form aliquid pharmaceutical preparation, or the acids, either singly or inmixtures thereof, can be combined with a solid, non-toxic,orallyingestible, pharmaceutical carrier to form a solid preparationsuch as a tablet or capsule. The pharmaceutical preparations may besterilized or contain auxiliaries such as preserving, stabilizing,wetting, emulsifying or dispersing agents, disintegrating agents andlubricating agents. The compositions either in liquid form or solid formare preferably formulated to contain between 50 mg. and mg. of the aboveglutamic acids, either singly or in mixtures thereof, per shaped dosageunit.

The following examples illustrate the invention. Unless indicated, partsare by weight.

EXAMPLE 9 50 parts of N-acetyl-u-L-aspartyl-L-glutamic acid are combinedwith 20 parts of lactone and 27 parts of a starch paste are added. Themixture is dried and pressed through a 20-mesh screen. 3 parts magnesiumstearate as lubricant are added and the granules are pressed into 100mg. tablets suitable for administration.

EXAMPLE 10 Tablets are prepared that have 50 mg, of active substance ineach tablet and have the following composition Mg.N-acetyl-a-L-aspartyl-L-glutamic acid 50 Lactone 20 Starch paste 27Magnesium stearate 3 Tablets may be formulated so that the daily dose ofglutamic acid amounts to 50 to 200 mg, preferably with 4 administrationsper day.

Toxicological data For both rats and mice, LD by mouth has been found tobe higher than 5000 mg./kg.

LD intravenously, for rats, was 292 mg./k g.

Intraperitoneally, doses of 0.5, 1 and 1.5 mg./kg. of body weight wereinjected to rats. The mortality, within 72 hours, was 0/6, 1/4 and 3/4(dead/treated). The symptoms were: visceral irritation, torpidity ofmotion and ataxia. Substantially, a toxic sedative state.

Rabbits were intracisternally treated with doses of 1, 2, and 3 mg./kg.The injection was preceded by the subtraction of an equal volume ofcerebrospinal fluid, and was effected slowly to avoid irritationphenomena which are easy to take place. The first 2 doses injected eachto 4 animals, caused an increase of the muscular tone and the appearanceof tremors. These symptoms continued for more than 4 hours.

After 12 hours the animals appeared completely normal.

In the following 7 days, no irritation could be observed in the treatedanimals.

In experiments of chronic toxicity, carried out on rats, results showthat by administering 50 mg./kg. daily for 3 months, by mouth, nochanges of weight, of liver and kidney function and of blood erasis areobserved.

No damage of the organs were detected during the autoptic examination ofthe treated animals.

For humans, the following was observed:

N-acetyl-aspartyl-glutamic acid, administered for 30 days in the form oftablets at the daily dose of 200 mg. divided in 4 doses of 50 mg. each,to subjects in good health conditions, provoked no intolerancephenomena. In fact, no nausea, vomiting, pyrosis or rash, diarrhea orconstipation phenomena were observed; the liver and kidney functionswere normal, and no changes occurred for the cardio-circulatoryapparatus.

7 Effects An investigation has been carried out on the effects of theadministration of N-acetyl-B-aspartybglutamic acid (NAAGA) by mouth tohumans, as to improvement in mental efficiency or to reduce mentalfatigue and related syndromes measured by means of some psychologicaltests.

Materials and methods Treated subjects.-40 individuals, a homogeneousgroup as to age (2040), sex (male) and education (from end of elementaryschool to three years general education thereafter) were utilized. The40 subjects were divided into two groups (A and B), every one comprisingindividuals selected at random.

To every one of the 20 components of group A, 4 tablets of placebo,equal as to form, color, odor and taste to the NAAGA tablets, wereadministered; two to three hours after the tablet administration theindividuals were subjected to the psychological tests. The next day,every individual received 4 or more placebo tablets and after two tothree hours the same psychological tests were practiced again.

The 20 subjects comprising group B received 4 placebo tablets each onthe first day, and two to three hours thereafter they were subjected tothe different psychological tests. On the following day there wereadministered 4 tablets containing 50 mg. NAAGA each, per individual, andthe psychological tests were repeated after twothree hours.

The rather complex experimental schedule, as above indicated, wasthought to be necessary in order to distinguish the eventual effects, onthe psychological tests, which could be ascribed to NAAGA, from thosewhich were ascribed to learning; that is to the tests improvement duemerely to their repetition.

Neither the treated subjects nor the examiners knew whether NAAGA orplacebo had been administered.

The 4 placebo tablets and the NAAGA tablets were swallowed all together.

Psychological tests The psychological tests were as follows:

(1) Cattel test (a reagent of general culture free intelligence by R. B.Cattel and A. K. S. Cattel). Scale 2 was used for adults with educationinferior to three years after elementary school, in its two parallelforms (one of which was employed on the first day of experiments, andthe other on the following day). This test measures especially theability to reason and evidences operations of the synthetical thought inthe 4-sub-tests comprised by each form. The evaluation is made on thebasis of total rating, that is to say, to the sum of ratings reported inthe sub-tests. A higher rating indicates a better result.

(2) Kohs cubes.-Two parallel series were used (one employed on the firstday and the other on the following day), each composed of eight coloreddesigns, to be reconstituted with equally colored cubes. This testaffords a measure of practical intelligence-that is to say, ofintellective qualities for the solution of practical problems. Theresult is expressed by the total rating as obtained according to thetime employed for reconstituting the eight designs of each series. Alsoin this test, a higher rating indicates a better result.

(3) Couve test.-This test consists of a picture containing the figuresfrom 1 to 100, in disorder, which the subject has to count aloudaccording to their natural progressions, without omitting any of thefigures. This test indicates the value of the attention qualities of aconactive type (imposed attention), Within a rather wide perceptivefield, with selection involving numerical data. The evaluation isexpressed by the last number found within 5 minutes.

(4) Memory.-Two parallel charts were used (one employed the first day ofthe experiments, the other one on the following day). containing 12different images each; the subject is allowed to observe the images for30 seconds and after 5 minutes he should try to reproduce them by meansof small image reproductions, to be selected among 25 and to be put on asuitable modulus. Thus, two aspects of memory are measured: the visibleone, strictly connected to the fixation of engrams (also according tosensorial efficiency), as well as to recalling the same engrams, and thetopographical one, relative to spacial localization of mnemonic images.The evaluation is made for visible memory on the basis of total, exactlyrecalled elements, and for topographical memory on the basis of total,correctly located elements.

(5) Thurstone scale.-This consists of a list of nine terms (nearlyexhaustedfatiguedprostrated-a little tired-neither tired norfatiguednearly well-in due forrn-wellvery well) among which the subject,at the end of the experiments, is invited to choose that which he thinkscorresponds most nearly to his state of fatigue.

This expresses the subjective fatigue, that is, the per ception whichthe subject has of his own general efficiency at the end of theexaminations. The nine terms are indicated, in the order as they arelisted above, by the figures 1 to 9, so that the higher figurescorrespond to a lower fatigue.

Results Table A shows the results relative to the 20 subjects of GroupA, who were subjected, as pointed out above, to the psychlological testsfor two consecutive days, the first day after receiving 4 placebotablets and the following day after receiving 4 placebo tablets again.

Table B shows, on the contrary, the results as obtained in the 20individuals of Group B, subject, as already stated to the psychologicaltests also for two consecutive days, however, the first day after beingadministered with 4 placebo tablets and the following day afteradministration of 4 NAAGA tablets.

In the group corresponding to table A, no considerable differences werefound between the results obtained the first day and those of thefollowing day. Some improvement-to be ascribed to learning-was notedonly in the Kohs cubes test (1st day: average 31.00; 2nd day, average34.85) and in the topographical memory (1st day; average 2.55; 2nd day,average 4.45). The other tests (Cattel, Couve, visible memory, Thurstonescale) gave almost identical results.

After NAAGA administration (table B), the Couve test-showed nonoteworthy variations (1st day, that is, after the placebo treatment;average 31.60; 2nd day, that is after the NAAGA treatment; 32.15).Visible memory also appeared scarely modified (1st day, average 7.93;2nd day, average 5.25). Topographical memory is improved (1st day,average 3.80; 2nd day, average 4.86), but an analogous variationoccurred, as it was already stressed, also in the control subjects(Table A) treated solely with placebo (1st day, average 2.55; 2nd day,average 4.45) and it is therefore to be ascribed to learnmg.

The improvement obtained after NAAGA in the Cattel test and in the Kohscubes test was, on the contrary, sharper.

In fact, while in the control subjects (Table A), the Cattel testremained unchanged (1st day, average 25.55; 2nd day, average 25.90), inthose treated with NAAGA it shows an average equal to 22.55 in the 1stday (that is after placebo administration) and 24.35 in the 2nd day,(tfha ty is after NAAGA administration), with an increase 0 8 0.

Still more sensible was the difference in the results relative to theKohs cubes test. In the control subjects (Table A) an increase of 12%(1st day, average 31.00; 2nd day, average 34.85) was recorded; in theindividuals treated with NAAGA the increase was by 25.5% (1st day,average 29.80; and 2nd day average 37.45).

TABLE A.SUBJECTS TREATED WITH PLACEBO Memory A Cattel Cubes CouveVisible Topographical Thurstone scale years I II I II I II I II II IAverage 25. 55 25. 90 31. 00 34 85 32. 50 33. 80 8. 8. 45 2. 55 4. 456.10 5. 5O

I=first day (after 4 placebo tablets). II=second day (after 4 placebotablets).

TABLE B.SUBJECTS TREATED WITH NAAGA Memory A Oattel ubes Couvo VisibleTopographical Thurstone scale years I II I II I II I II I II I IIAverage 22. 24. 35 29. 80 37. 45 60 32.15 7. 93 8. 26 3. 4. 86 '4. 40 5.25

I=first day (after 4 placebo tablets).

=second day (after 4 NAAGA tablets).

=The memory test was not carried out because of technical reasons.

What is claimed is:

1. A method for treating mental fatigue in man and animals whichcomprises internally administering to said man and animals atherapeutically effective amount of a member selected from the groupconsisting of:

N-acetyl-a-L-aspartyl-L-glutamic acid,

N-acetyl-fi-L-aspartyl-L-g1utamic acid.

2. A method as claimed in claim 1 wherein the member isN-acetyl-a-L-aspartyl-L-glutamic acid.

3. A method as claimed in claim 1 where the member isN-acetyl-B-L-aspartyl-L-glutamic acid.

4. A method as claimed in claim 1 wherein said therapeutically eifectiveamount is 50 mg. to mg.

5. A method as claimed in claim 1 wherein the member is administered inthe form of a solid pharmaceutical composition in shaped dosage unit fororal ingestion; the said dosage unit containing at least 10 mg. of saidmember and a solid, nontoxic, orally-ingestible, pharmaceutical carrier.

6. A method as claimed in claim 5 wherein the com- 5 pound isN-acetyl-a-L-aspartyl-L-glutamic acid.

OTHER REFERENCES C-hem. Abst. 64-6744e (1966). Science, vol. 108, p. 281(Sept. 10, 1948).

STANLEY J. FRIEDMAN, Primary Examiner

